L’uranium (U) est naturellement présent à l’état de trace dans l’eau (µg.L-1), sa concentration pouvant atteindre localement quelques mg.L-1 du fait des activités anthropiques. Plusieurs études écotoxicologiques sur Danio rerio ont mis en évidence la toxicité, e.g. le stress oxydant, la génotoxicité mais aussi la reprotoxicité (i.e. moins de pontes et d’œufs pondus chez les poissons contaminés) de l’U dont les mécanismes ne sont pas connus.L’objectif de cette étude est de contribuer à la compréhension de la reprotoxicité de l’U par l’élucidation de mécanismes moléculaires perturbés après contamination. Pour cela, des investigations ont été menées sur les ovaires de poissons zèbre Danio rerio, reproduits (R) ou non (NR), après exposition par voie directe en condition de laboratoire à des concentrations représentatives d’environnements contaminés.Ce travail de thèse a été divisé en deux volets. Un premier volet analytique avait pour but la poursuite des développements de méthodes pour l’identification des complexes U-protéines en condition non dénaturante, autour du couplage de techniques de séparation (chromatographie d’exclusion stérique SEC, électrophorèse hors gel OGE) et de détection sensible par spectrométrie de masse élémentaire (ICP MS) et moléculaire (ESI MS). Le second volet a été dédié à l’étude de la reprotoxicité de l’U à l’échelle moléculaire, avec i) l’étude des complexes natifs U-protéines (approche métallomique), et ii) l’analyse différentielle de l’expression des protéines (approche protéomique).Les développements analytiques ont permis de garder le tampon physiologique et non dénaturant d’extraction pour l’étape de séparation OGE, améliorant le taux de recouvrement en U. En écotoxicologie, les principaux résultats montrent que l’ovaire est un organe accumulateur de l’U et que le statut de reproduction a une influence sur le niveau d’accumulation (R<NR). En revanche, cet état a peu d’influence sur sa distribution protéique pour laquelle 4 fractions (dont 1 principale) ont été identifiées, toutes contenant aussi du phosphore. L’identification des cibles potentielles de l’U et des protéines exprimées différentiellement (vtg, GST, GAPDH,…) a montré que les processus biologiques perturbés suite à la contamination sont de deux niveaux : 1/ générique (stress oxydant) et 2/ plus spécifique de la gonade (développement et maturation des ovocytes). En conclusion, ces deux approches complémentaires ont permis de mettre en évidence un effet direct (complexation) et indirect (expression protéique modulée) de l’U, et de proposer l’hypothèse d’un défaut de maturation des ovocytes après contamination. Ce défaut pourrait impacter le développement embryonnaire et in fine expliquer la reprotoxicité observée lors d’études écotoxicologiques précédentes. / Uranium (U) is naturally presents at trace level (µg.L-1) in aquatic environment; its concentration can increase up to a few mg.L-1 due to human activities. Several ecotoxicological studies have shown uranium toxicity in contaminated zebrafishes Danio rerio, e.g. oxidative stress, genotoxicity and reprotoxicity (i.e. lower number of spawn and eggs laid) but mechanisms are not well known.The objective of this study is to contribute to the understanding of uranium reprotoxicity by elucidating the disrupted molecular mechanisms after contamination. Therefore, investigations have been carried out on ovaries from reproduced (R) and non-reproduced (NR) zebrafishes after waterborne exposure in laboratory conditions at environmentally relevant concentrations.This project was divided into two parts. Firstly, analytical investigations were carried out to continue the development of non-denaturing methods for U-protein identification by coupling separative techniques (size exclusion chromatography SEC, off gel electrophoresis OGE) with elemental (ICP MS) and molecular (ESI MS) sensitive mass spectrometry detection. Secondly, studies of U reprotoxicity were investigated by studying i) native U-protein complexes (metallomics approach) and ii) differential analysis of protein expression (proteomics approach)Analytical developments allowed keeping the physiological and non-denaturing extraction buffer for OGE separation step, improving U recovery. In ecotoxicology, the major results showed that ovary is an U accumulating organ and that the reproduction status modifies the accumulation level (R<NR). However, this status is of little influence on its distribution on proteins with 4 fractions (including a major one) determined, all of them coeluting with phosphorus. The identification of U potential targets and of protein expression differences (vtg, GST, GAPDH…) showed that biological processes disrupted after contamination are at two levels: 1/ generic (oxidative stress) and 2/ more specific to gonad (oocyte development and maturation).As a conclusion, these two complementary approaches showed a direct (complexation) and indirect (modification of protein expression) effects of U, and enabled to hypothesize a lack of oocyte maturation after contamination. This defect could impact embryo development and in fine explain the reprotoxicity observed in previous ecotoxicological studies.

A cellular pre-mRNA undergoes various post-transcriptional processing events, including capping, splicing and polyadenylation prior to nuclear export. Splicing is particularly important for mRNA nuclear export as two distinct multi-protein complexes, known as human TREX (hTREX) and the exon-junction complex (EJC), are recruited to the mRNA in a splicing-dependent manner. In contrast, a number of Kaposi's sarcoma-associated herpesvirus (KSHV) lytic mRNAs lack introns and are exported by the virus-encoded ORF57 protein. Herein we show that ORF57 binds to intronless viral mRNAs and functions to recruit the complete hTREX complex, but not the EJC, in order assemble an export component viral ribonucleoprotein particle (vRNP). The formation of this vRNP is mediated by a direct interaction between ORF57 and the hTREX export adapter protein, Aly. Aly in turn interacts directly with the DEAD-box protein UAP56, which functions as a bridge to recruit the remaining hTREX proteins to the complex. Moreover, we show that a point mutation in ORF57 which disrupts the ORF57-Aly interaction leads to a failure in the ORF57-mediated recruitment of the entire hTREX complex to the intronless viral mRNA and inhibits the mRNAs subsequent nuclear export and virus replication. Furthermore, we have utilised a trans-dominant Aly mutant to prevent the assembly of the complete ORF57-hTREX complex; this results in a vRNP consisting of viral mRNA bound to ORF57, Aly and the nuclear export factor, TAP. Strikingly, although both the export adapter Aly and the export factor TAP were present on the viral mRNP, a dramatic decrease in intronless viral mRNA export and virus replication was observed in the absence of the remaining hTREX components (UAP56 and hTHO-complex). Together, these data provide the first direct evidence that the complete hTREX complex is essential for the export of KSHV intronless mRNAs and infectious virus production.

Molecular Biology has allowed the characterization and manipulation of the molecules of life in the wet lab. Also the structures of those macromolecules are being continuously elucidated. During the last decades of the past century, there was an increasing interest to study how the different genes are organized into different organisms (‘genomes’) and how those genes are expressed into proteins to achieve their functions. Currently the sequences for many genes over several genomes have been determined. In parallel, the efforts to have the structure of the proteins coded by those genes go on. However it is experimentally much harder to obtain the structure of a protein, rather than just its sequence. For this reason, the number of protein structures available in databases is an order of magnitude or so lower than protein sequences. Furthermore, in order to understand how living organisms work at molecular level we need the information about the interaction of those proteins. Elucidating the structure of protein macromolecular assemblies is still more difficult. To that end, the use of computers to predict the structure of these complexes has gained interest over the last decades.<p>The main subject of this thesis is the evaluation of current available computational methods to predict protein – protein interactions and build an atomic model of the complex. The core of the thesis is the evaluation protocol I have developed at Service de Conformation des Macromolécules Biologiques et de Bioinformatique, Université Libre de Bruxelles, and its computer implementation. This method has been massively used to evaluate the results on blind protein – protein interaction prediction in the context of the world-wide experiment CAPRI, which have been thoroughly reviewed in several publications [1-3]. In this experiment the structure of a protein complex (‘the target’) had to be modeled starting from the coordinates of the isolated molecules, prior to the release of the structure of the complex (this is commonly referred as ‘docking’).<p>The assessment protocol let us compute some parameters to rank docking models according to their quality, into 3 main categories: ‘Highly Accurate’, ‘Medium Accurate’, ‘Acceptable’ and ‘Incorrect’. The efficiency of our evaluation and ranking is clearly shown, even for borderline cases between categories. The correlation of the ranking parameters is analyzed further. In the same section where the evaluation protocol is presented, the ranking participants give to their predictions is also studied, since often, good solutions are not easily recognized among the pool of computer generated decoys.<p>An overview of the CAPRI results made per target structure and per participant regarding the computational method they used and the difficulty of the complex. Also in CAPRI there is a new ongoing experiment about scoring previously and anonymously generated models by other participants (the ‘Scoring’ experiment). Its promising results are also analyzed, in respect of the original CAPRI experiment. The Scoring experiment was a step towards the use of combine methods to predict the structure of protein – protein complexes. We discuss here its possible application to predict the structure of protein complexes, from a clustering study on the different results.<p>In the last chapter of the thesis, I present the preliminary results of an ongoing study on the conformational changes in protein structures upon complexation, as those rearrangements pose serious limitations to current computational methods predicting the structure protein complexes. Protein structures are classified according to the magnitude of its conformational re-arrangement and the involvement of interfaces and particular secondary structure elements is discussed. At the end of the chapter, some guidelines and future work is proposed to complete the survey. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

The fidelity of biological processes and reactions, inspite of the widespread diversity, is programmed by highly specific physico-chemical principles. This underlines our basic understanding of different interesting phenomena of biological relevance, ranging from enzyme specificity to allosteric communication, from selection of fold to structural organization / states of oligomerization, from half-sites-reactivity to reshuffling of the conformational free energy landscape, encompassing the dogma of sequence-structure dynamics-function of macromolecules. The role of striking an optimal balance between rigidity and flexibility in macromolecular 3D structural organisation is yet another concept that needs attention from the functional perspective. Needless to say that the variety of protein structures and conformations naturally leads to the diversity of their function and consequently many other biological functions in general. Classical models of allostery like the ‘MWC model’ or the ‘KNF model’ and the more recently proposed ‘population shift model’ have advanced our understanding of the underlying principles of long range signal transfer in macromolecules. Extensive studies have also reported the importance of the fold selection and 3D structural organisation in the context of macromolecular function. Also ligand induced conformational changes in macromolecules, both subtle and drastic, forms the basis for controlling several biological processes in an ordered manner by re-organizing the free energy landscape. The above mentioned biological phenomena have been observed from several different biochemical and biophysical approaches. Although these processes may often seem independent of each other and are associated with regulation of specialized functions in macromolecules, it is worthwhile to investigate if they share any commonality or interdependence at the detailed atomic level of the 3D structural organisation. So the nagging question is, do these diverse biological processes have a unifying theme, when probed at a level that takes into account even subtle re-orchestrations of the interactions and energetics at the protein/nucleic acid side-chain level. This is a complex problem to address and here we have made attempts to examine this problem using computational tools. Two methods have been extensively applied: Molecular Dynamics (MD) simulations and network theory and related parameters. Network theory has been extensively used in the past in several studies, ranging from analysis of social networks to systems level networks in biology (e.g., metabolic networks) and have also found applications in the varied fields of physics, economics, cartography and psychology. More recently, this concept has been applied to study the intricate details of the structural organisation in proteins, providing a local view of molecular interactions from a global perspective. On the other hand, MD simulations capture the dynamics of interactions and the conformational space associated with a given state (e.g., different ligand-bound states) of the macromolecule. The unison of these two methods enables the detection and investigation of the energetic and geometric re-arrangements of the 3D structural organisation of macromolecule/macromolecular complexes from a dynamical or ensemble perspective and this has been one of the thrust areas of the current study. So we not only correlate structure and functions in terms of subtle changes in interactions but also bring in conformational dynamics into the picture by studying such changes along the MD ensemble. The focus was to identify the subtle rearrangements of interactions between non-covalently interacting partners in proteins and the interacting nucleic acids. We propose that these rearrangements in interactions between residues (amino acids in proteins, nucleic acids in RNA/DNA) form the common basis for different biological phenomena which regulates several apparently unrelated processes in biology. Broadly, the major goal of this work is to elucidate the physico-chemical principles underlying some of the important biological phenomena, such as allosteric communication, ligand induced modulation of rigidity/flexibility, half-sites-reactivity and so on, in molecular details. We have investigated several proteins, protein-RNA/DNA complexes to formulate general methodologies to address these questions from a molecular perspective. In the process we have also specifically illuminated upon the mechanistic aspects of the aminoacylation reaction by aminoacyl-tRNA synthetases like tryptophanyl and pyrrolysyl tRNA synthetase, structural details related to an enzyme catalyzed reaction that influences the process of quorum sensing in bacteria. Further, we have also examined the ‘dynamic allosterism’ that manipulates the activity of MutS, a prominent component of the DNA bp ‘mismatch repair’ machinery. Additionally, our protein structure network (PSN) based studies on a dataset of Rossmann fold containing proteins have provided insights into the structural signatures that drive the adoption of a fold from a repertoire of diverse sequences. Ligand induced percolations distant from the active sites, which may be of functional relevance have also been probed, in the context of the S1A family of serine proteases. In the course of our investigation, we have borrowed several concepts of network parameters from social network analysis and have developed new concepts. The Introduction (Chapter-1) summarizes the relevant literature and lays down a suitable background for the subsequent chapters in the thesis. The major questions addressed and the main goal of this thesis are described to set an appropriate stage for the detailed discussions. The methodologies involved are discussed in Chapter-2. Chapter-3 deals with a protein, LuxS that is involved in the bacterial quorum sensing; the first part of the chapter describes the application of network analysis on the static structures of several LuxS proteins from different organisms and the second part of this chapter describes the application of a dynamic network approach to analyze the MD trajectories of H.pylori LuxS. Chapter-4 focuses on the investigation of human tryptophanyl-tRNA synthetase (hTrpRS), with an emphasis to identify ligand induced subtle conformational changes in terms of the alternation of rigidity/flexibility at different sites and the re-organisation of the free energy landscape. Chapter-5 presents a novel application of a quantum clustering (QC) technique, popular in the fields of pattern recognition, to objectively cluster the conformations, sampled by molecular dynamics simulations performed on different ligand bound structures of the protein. The protein structure network (PSN) in the earlier studies were constituted on the basis of geometric interactions. In Chapters 6 and 7, we describe the networks (proteins+nucleic acids) using interaction energy as edges, thus incorporating the detailed chemistry in terms of an energy-weighted complex network. Chapter-6 describes an application of the energy weighted network formalism to probe allosteric communication in D.hafniense pyrrolysyl-tRNA synthetase. The methodology developed for in-depth study of ligand induced changes in DhPylRS has been adopted to the protein MutS, the first ‘check-point protein’ for DNA base pair (bp) mismatch repair. In Chapter-7, we describe the network analysis and the biological insights derived from this study (the work is done in collaboration with Prof. David Beveridge and Dr. Susan Pieniazek). Chapter-8 describes the application of a network approach to capture the ligand-induced subtle global changes in protein structures, using a dataset of high resolution structures from the S1A family of serine proteases. Chapter-9 deals with probing the structural rationale behind diverse sequences adopting the same fold with the NAD(P)-binding Rossmann fold as a case study. Future directions are discussed in the final chapter of the thesis (Chapter-10).

Die Schwammskelette der Ordnung Verongida zeichnen sich durch das Fehlen mineralischer Komponenten aus. Stattdessen bestehen sie aus Spongin, einem kollagenartigen Protein, und Chitin. Im Rahmen der vorliegenden Arbeit wurden die aus solch einem Chitin-Protein-Komplex bestehenden Skelette der Schwammspezies Aplysina cavernicola und Ianthella basta aus der Ordnung Verongida untersucht. Aufgrund ihrer morphologischen Unterschiede wurde für jede Schwammart eine eigene Methode zur Isolierung der Schwammskelette entwickelt. Die isolierten Skelette konnten anschließend mit verschiedenen Methoden wie REM, ATR-FTIR-Spektroskopie und NMR-Spektroskopie charakterisiert werden. Weiterhin wurde eine Methode zur Extraktion und Analyse der in den Skeletten befindlichen Aminosäuren mittels GC-MS entwickelt. Die Untersuchungen zeigten deutlich, dass es sich bei Spongin um ein kollagenartiges und halogeniertes Protein handelt, welches je nach Schwammart Unterschiede in der Aminosäurezusammensetzung aufweist. Darüber hinaus gelang es zum ersten Mal das Chitin aus dem Chitin-Protein-Komplex mittels Phosphorsäure zu entfernen. Aus den gewonnenen Erkenntnissen konnte abschließend ein Modell zum Aufbau des Chitin-Protein-Komplexes in der Schwammspezies Aplysina cavernicola entwickelt werden.

L'activació sostinguda de Hog1 porta a una inhibició del creixement cel·lular. En aquest treball, hem observat que el fenotip de letalitat causat per l'activació sostinguda de Hog1 és parcialment inhibida per la mutació del complexe SCFCDC4. La inhibició de la mort causada per l'activació sostinguda de Hog1 depèn de la via d'extensió de la vida. Quan Hog1 s'activa de manera sostinguda, la mutació al complexe SCFCDC4 fa que augmenti l'expressió gènica depenent de Msn2 i Msn4 que condueix a una sobreexpressió del gen PNC1 i a una hiperactivació de la deacetilassa Sir2. La hiperactivació de Sir2 és capaç d'inhibir la mort causada per l'activació sostinguda de Hog1. També hem observat que la mort cel·lular causada per l'activació sostinguda de Hog1 és deguda a una inducció d'apoptosi. L'apoptosi induïda per Hog1 és inhibida per la mutació al complexe SCFCDC4. Per tant, la via d'extensió de la vida és capaç de prevenir l'apoptosi a través d'un mecanisme desconegut. / Sustained Hog1 activation leads to an inhibition of cell growth. In this work, we have observed that the lethal phenotype caused by sustained Hog1 activation is prevented by SCFCDC4 mutants. The prevention of Hog1-induced cell death by SCFCDC4 mutation depends on the lifespan extension pathway. Upon sustained Hog1 activation, SCFCDC4 mutation increases Msn2 and Msn4 dependent gene expression that leads to a PNC1 overexpression and a Sir2 deacetylase hyperactivation. Then, hyperactivation of Sir2 is able to prevent cell death caused by sustained Hog1 activation. We have also observed that cell death upon sustained Hog1 activation is due to an induction of apoptosis. The apoptosis induced by Hog1 is decreased by SCFCDC4 mutation. Therefore, lifespan extension pathway is able to prevent apoptosis by an unknown mechanism.